Application of mogroside V in preparation of medicine for dispelling effects of alcohol and protecting liver

Abstract

The invention relates to the field of medicines, and particularly discloses application of mogroside V in preparation of a medicine for dispelling the effects of alcohol and protecting the liver, experiments prove that the anti-alcohol effect of the mogroside V is obviously superior to that of a mogroside crude extract, and in one embodiment, the mogroside V can increase protein expression of ethanol dehydrogenase and acetaldehyde dehydrogenase in the liver of a mouse at the same time. In one embodiment, the mogroside V can prolong the drunkenness tolerance time of a mouse and shorten the drunkenness time of the mouse. In another embodiment, the mogroside V can relieve liver inflammation caused by alcohol. In a further embodiment, the mogroside V can greatly relieve liver fat deposition caused by alcohol. Therefore, the mogroside V can be developed into a brand new product for dispelling the effects of alcohol and protecting the liver, and has a huge application prospect.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the application of mogroside V. Background Art

[0002] Long-term drinking leads to alcoholic liver disease (ALD) such as alcoholic fatty liver disease and alcoholic hepatitis. In many countries, ALD is one of the main causes of end-stage liver disease and the need for liver transplantation. Therefore, innovative preventive measures and treatments are urgently needed to address this growing health problem. In recent years, research on natural products and their derivatives in preventing and alleviating the intoxication state has shown good prospects.

[0003] Momordica grosvenori (scientific name Siraitia grosvenorii (Swingle) C. Jeffrey ex Lu et ZYZhang) is the dried fruit of the perennial herbaceous vine of the Cucurbitaceae family, the genus Momordica, mainly produced in Guilin, Guangxi. According to traditional Chinese medicine, Momordica grosvenori is sweet and cool in nature, and belongs to the lung and large intestine meridians. It has the effects of moistening the lungs and relieving coughs, promoting fluid production and relieving thirst, and moistening the intestines and relieving constipation. The folk medicinal history of Momordica grosvenori has been more than 300 years. It is one of the first batch of approved Chinese medicines for both medicinal and edible purposes. It has therapeutic effects on tuberculosis, hypertension, gastritis, asthma, whooping cough, acute and chronic tracheitis, constipation, etc.

[0004] The unique ingredients in Luo Han Guo, mogrosanol and its derivatives, mogrosan saponins, are a class of cucurbitane-type triterpene glycosides, among which mogrosan saponins are cucurbitane-type triterpene glycosides, and their aglycones are mogrosanol. Mogrosan saponins are a class of chemical components that have been studied more frequently in Luo Han Guo.

[0005] At present, the cucurbitane-type triterpenoid components extracted, separated and identified from Momordica grosvenori fruit include mogroside alcohol, mogroside IIA1, mogroside IIE, mogroside III, mogroside IIIE, mogroside IV, mogroside IVA, mogroside IVE, mogroside V, mogroside VI, 11-oxo-mogroside alcohol, 11-oxo-mogroside V, etc. The crude extract of mogroside has been reported to have the effect of sobering up.

[0006] Another extract of monk fruit is mogroside V, which is widely used as a sweetener in beverages due to its low calorie content and extremely high sweetness, about 300 times that of sucrose. Currently, there are reports that mogroside V has multiple pharmacological activities, including anti-inflammatory, antioxidant, anti-cancer and hypoglycemic effects. Summary of the invention

[0007] The chemical structure of mogroside V is as follows:

[0008] In order to develop a new alcohol sobering, hangover sobering and liver protection drug, the inventors studied and screened a wide variety of mogrosides one by one, and found that mogroside V can stimulate the activity and protein expression of alcohol dehydrogenase and acetaldehyde dehydrogenase to play a role in sobering up. At the same time, it was found that mogroside V can downregulate the expression of cellular inflammatory factors caused by alcohol and upregulate the expression of lipid metabolism-related proteins in the liver, thereby reducing alcohol-induced liver inflammation and liver fat deposition, and playing a liver protection role.

[0009] The inventors found through mouse experiments that the effect of mogroside V in reducing the blood alcohol content of drinking mice is significantly better than that of sweet and sweet crude extract of mogroside. Mogroside V can increase the activity and protein expression of alcohol dehydrogenase and acetaldehyde dehydrogenase in the liver of drinking mice; Mogroside V can prolong the tolerance time of drunkenness in mice and shorten the drunkenness time of mice; Mogroside V can alleviate alcohol-induced liver lesions; Mogroside V reduces alcohol-induced hepatic fat deposition.

[0010] Therefore, the present invention discloses the application of mogroside V in preparing alcohol-relieving and liver-protecting medicines.

[0011] Furthermore, the mogroside V can alleviate liver inflammation caused by alcohol.

[0012] Furthermore, the mogroside V can reduce liver fat deposition caused by alcohol.

[0013] Furthermore, the mogroside V is used alone or in combination with Chinese medicinal materials or chemical drugs having a sobering effect to prepare a sobering and liver-protecting drug.

[0014] Furthermore, the mogroside V is combined with a pharmaceutically acceptable carrier to prepare an oral preparation.

[0015] Furthermore, the oral preparation is a tablet, capsule, granule, spray, lozenge, pill or oral liquid.

[0016] The invention discloses a new use of mogroside V, which is used for preparing drugs for sobering up, sobering up and protecting the liver. In addition, it has the advantages of being safe, effective, having no side effects, and having a good taste, and can be used as a new product for sobering up, sobering up and protecting the liver, thereby meeting people's needs for sobering up and protecting the liver, and solving various problems caused by excessive drinking. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a schematic diagram of how Mogroside V in Example 1 reduces the ethanol content in the blood of mice.

[0018] Figure 2is the alcohol tolerance time of mice on mogroside V in Example 2 ( Figure 2 A) and the effect of drunkenness time ( Figure 2 B).

[0019] Figure 3 This is a graph showing the effect of Mogroside V on the activity of enzymes related to ethanol metabolism in the liver of mice in Example 3; in, Figure 3 A is the result of alcohol dehydrogenase test; Figure 3 B is the result of acetaldehyde dehydrogenase test.

[0020] Figure 4 This is a graph showing the effect of Mogroside V on the expression of enzyme proteins related to ethanol metabolism in mouse liver in Example 3; in, Figure 4 A is the protein immunoblot of CYP2E1, ADH1, and ALDH2 in mouse liver; Figure 4 B is the quantification of CYP2E1 protein immunoblot in mouse liver; Figure 4 C is the quantification of ADH1 protein immunoblotting in mouse liver; Figure 4 D is the quantification of ALDH2 protein immunoblotting in mouse liver.

[0021] Figure 5 This is a graph showing the effect of Mogroside V on the pathological state of mouse liver in Example 5.

[0022] Figure 6 This is a graph showing the effect of Mogroside V on fat deposition in mouse liver in Example 6. DETAILED DESCRIPTION

[0023] The present invention is further described below with reference to the embodiments and drawings, but is not intended to limit the present invention.

[0024] Example 1: Experiment on reducing ethanol content in the blood of mice by Mogroside V.

[0025] Animal treatment: 8-week-old C57BL / 6J male mice (20-23 g) were provided by Hunan Slake Jingda Laboratory Animal Co., Ltd. (Hunan, China). The chronic alcohol drinking plus binge alcohol feeding animal model of the National Institute on Alcohol Abuse and Alcoholism (NIAAA) was used to establish the animal model of alcoholic liver injury. Briefly, mice were randomly divided into five groups of five mice each after free access to Lieber-DeCarli liquid control diet (Biotech-HD, Beijing, China) for five days.

[0026] The specific dosing regimen is as follows: Blank group: liquid feed control, distilled water was given by gavage every day.

[0027] Model group: alcohol liquid feed, distilled water was given by gavage every day.

[0028] Siraitia grosvenori crude extract group: alcohol liquid feed, daily oral administration of 100 mg / kg Siraitia grosvenori crude extract aqueous solution.

[0029] Mogroside V group: alcohol liquid feed, daily oral administration of 100 mg / kg Mogroside V aqueous solution.

[0030] Mice fed with alcohol liquid diet were fed with Lieber-DeCarli liquid diet containing 5% (V / V) ethanol for 10 days, while the blank group received an isocaloric control diet. Starting from the 6th day, the assigned drugs or saline were given by gavage at 9 am every day. On the 16th day, 1 hour after drug administration, the mice in the model group were given 31.5% ethanol (5 g / kg) in the early morning, while the control group was given isocaloric maltodextrin half an hour before the corresponding drugs or saline by gavage. After fasting for 9 hours, all mice were euthanized, and blood samples and liver tissues were collected. 50 mg of liver tissue was mixed with 450 μl of saline under ice-cold conditions to prepare homogenates. Then, the homogenates were centrifuged at 4000 rpm at 4°C for 10 minutes, and the supernatant was carefully collected for the detection of liver biochemical indicators.

[0031] After 1.5 hours of ethanol gavage, the mice plasma samples were collected and the ethanol content in the plasma was detected by a kit. Figure 1 The results showed that both the crude extract of Momordica grosvenori and Momordica saponin V could reduce the ethanol content in the blood of mice, and the effect of Momordica saponin V was significantly better than that of the crude extract of Momordica grosvenori.

[0032] Example 2: Experiment on the effect of Mogroside V on alcohol tolerance time and drunkenness time in mice.

[0033] Animal treatment: 8-week-old C57BL / 6J male mice (20–23 g) were provided by Hunan Slake Jingda Laboratory Animal Co., Ltd. (Hunan, China). The chronic alcohol drinking plus binge alcohol feeding animal model of the National Institute on Alcohol Abuse and Alcoholism (NIAAA) was used to induce alcoholic liver injury. Briefly, mice were randomly divided into five groups of three mice each after free access to Lieber-DeCarli liquid control diet (Biotech-HD, Beijing, China) for five days.

[0034] The specific dosing regimen is as follows: Blank group: liquid feed control, distilled water was given by gavage every day.

[0035] Model group: alcohol liquid feed, distilled water was given by gavage every day.

[0036] Low-dose group of Mogroside V: alcohol liquid feed, daily oral administration of 50 mg / kg Mogroside V aqueous solution.

[0037] High-dose group of Mogroside V: alcohol liquid feed, daily oral administration of 100 mg / kg Mogroside V aqueous solution.

[0038] Positive control group: alcohol liquid feed, oral administration of 100 mg / kg silybin aqueous solution.

[0039] Mice fed with alcoholic liquid diet were fed with Lieber-DeCarli liquid diet containing 5% (V / V) ethanol for 10 days, while the blank group received an isocaloric control diet. Starting from the 6th day, the assigned drugs or saline were given by gavage at 9 am every day. On the 16th day, 1 hour after drug administration, mice in the model group, the monk fruit crude extract group, and the mogroside V group were given 31.5% ethanol (5 g / kg) in the early morning, while the blank group was given isocaloric maltodextrin. After fasting for 9 hours, all mice were euthanized and liver tissues were collected.

[0040] LORR (loss of righting reflex) test: After ethanol administration (time recorded as 0 minutes), the mice were observed. When the mice showed ataxia, they were placed on their backs on a V-shaped metal trough, and the time to reach LORR was recorded, which was recorded as the intoxication tolerance time. The time to recover from the intoxication state was also recorded, which was recorded as the intoxication time. LORR was defined as the inability to right the body from an inverted position three times within 30 seconds. For statistical analysis, if the alcohol tolerance time exceeded 30 minutes, it was counted as 30 minutes. If the sobriety time exceeded 300 minutes, it was not counted, indicating that the mouse could not recover from LORR.

[0041] Reference Figure 2 A, After oral administration of 31.5% ethanol solution, mice in the model group and low-dose group became drunk (lost righting reflex) in about 5 minutes, while the tolerance time of mice in the high-dose group and silybin group was significantly extended to 10-15 minutes.

[0042] Reference Figure 2 B, The drunkenness time of mice in the model group, low-dose group and silybin group was about 225 minutes, while the drunkenness time of mice in the high-dose group was significantly shortened to about 150 minutes. This indicates that mogroside V can not only prolong the alcohol tolerance time of mice, but also shorten the recovery time of drunkenness.

[0043] Example 3: Experiment on the effect of mogroside V on the activities of alcohol dehydrogenase and acetaldehyde dehydrogenase in mouse liver.

[0044] 50 mg of liver tissue was mixed with 450 μl of saline under ice-cold conditions to prepare homogenate. Then, the homogenate was centrifuged at 4000 rpm for 10 minutes at 4°C, and the supernatant was carefully collected for the detection of liver biochemical indicators. Alcohol dehydrogenase (A083-2-1) and acetaldehyde dehydrogenase (A075-1-1) were used to measure the activity of ethanol metabolism-related enzymes in mouse liver tissue.

[0045] Reference Figure 3 A, After being given liquid feed containing alcohol, the activity of alcohol dehydrogenase in the liver of mice in the model group was significantly reduced. Low doses of mogroside V and silybin could not effectively increase the activity of alcohol dehydrogenase, while high doses of mogroside V could significantly increase the activity of alcohol dehydrogenase.

[0046] Reference Figure 3 B. After being given liquid feed containing alcohol, the activity of acetaldehyde dehydrogenase in the liver of the model group mice was significantly reduced. Low-dose mogroside V could not effectively restore this downward trend, while high-dose group and silybin could significantly increase the activity of acetaldehyde dehydrogenase. This indicates that mogroside V significantly increased the activity of enzymes related to ethanol metabolism in the liver of mice.

[0047] Example 4: Experiment on the effect of Mogroside V on the expression of enzyme proteins related to ethanol metabolism in mice.

[0048] Protein expression of ethanol metabolism-related enzymes in mouse liver was determined by Western blotting: 1) Weigh 50 mg of frozen liver tissue, add PIPA lysis buffer (1 ml) to grind, place on ice for lysis for 30 minutes, centrifuge at 4°C, 12,000 rpm for 20 minutes, extract the supernatant as the total protein of liver tissue, quantify the protein concentration by BCA method, and denature the protein at high temperature; 2) Glue preparation: clean and dry the glass plate, assemble and fix it on the gel rack, mix the separation gel and pour it into the glass interlayer, add double distilled water to press the gel on the top of the liquid, let it stand at room temperature for 30 minutes, wash the top of the gel with double distilled water, and dry the top liquid with absorbent paper. Pour the concentrated gel, insert the comb, and let it stand at room temperature for 30 minutes; 3) Loading: adjust the protein concentration according to the results of protein quantification. After mixing the sample with the loading buffer, heat the protein to denature it, mix well and add it to the loading well. The protein loading amount of liver tissue is 15 μg. 4) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE): Add the sample to the electrophoresis well, set the voltage of the concentrated gel to 80V and the separation gel to 120V, and perform SDS-PAGE electrophoresis for 2 hours. Take out the gel and mark it; 5) Transfer: Prepare methanol-activated PVDF membrane, assemble the transfer sandwich in a plate containing transfer buffer, assemble in the order of filter paper, gel, membrane, filter paper, put the assembled sandwich into the transfer instrument, constant current 260mA, transfer for 90 minutes; 6) Blocking and elution: After labeling the PVDF membrane, transfer it to a blocking buffer containing 5% skim milk at room temperature for 3 hours, add PBST, and elute at room temperature with shaking for 10 minutes × 3 times; 7) Primary antibody incubation and elution: Add the corresponding primary antibody according to the required dilution, seal and incubate at 4°C overnight, remove the membrane, and rinse with PBST at room temperature for 10 minutes × 3 times; 8) Secondary antibody incubation and elution: Add diluted secondary antibody solution, shake at room temperature for 1 hour, and rinse with PBST for 10 minutes × 3 times; 9) ECL chemiluminescence development: immerse the membrane in the prepared ECL color developing solution (prepared before use) and expose it in the machine; 10) Image analysis: Save the image and use ImagJ to analyze the grayscale value.

[0049] The results of the effects of mogroside V on the expression of enzyme proteins related to ethanol metabolism in mice, such as Figure 4 shown.

[0050] Reference Figure 4 A, The expression of CYP2E1 in the liver of mice in the model group was significantly increased, and the expression of ADH1 and ALDH2 was significantly decreased. Compared with the model group, the expression of CYP2E1 in the high-dose group was significantly inhibited, and the expression of ADH1 and ALDH2 was significantly restored. This indicates that Mogroside V significantly increased the protein expression of ethanol metabolism-related enzymes in the liver of mice. The quantitative diagram of related protein expression content is shown in Figure 2. Figure 4 B, C and D.

[0051] Example 5: Experiment on the effect of mogroside V on the pathological state of mouse liver.

[0052] Histochemistry: The first step was paraffin section preparation and dewaxing. After the tissue surface was washed with PBS buffer, the mouse liver tissue was fixed with 10% neutral formalin and embedded in paraffin for sectioning. Step 2: Hematoxylin staining: Slice into hematoxylin solution for 3-5 minutes, rinse with running water, differentiate, rinse with running water, turn blue, rinse with running water; Step 3: Eosin staining: The sections were dehydrated in 85% and 95% graded alcohol for 5 minutes each, and then stained in eosin solution for 5 minutes. Step 4: dehydration, sealing, microscopic examination, image acquisition and analysis; Reference Figure 5In the model group, the hepatocytes of mice were arranged irregularly, the intercellular spaces were enlarged, the hepatic cord structure was destroyed, the fat vacuoles increased, and the inflammatory cells infiltrated. In the high-dose group and silybin group, the hepatocytes of mice were arranged regularly, the intercellular spaces were significantly reduced, the hepatic cord structure was relatively complete, and the number of fat vacuoles was significantly reduced. This indicates that mogroside V improves the effects of ethanol on the liver of mice.

[0053] Example 6: Experiment on the effect of mogroside V on fat deposition in mouse liver.

[0054] Oil Red O staining: In the first step, the frozen sections were placed in room temperature liver for 15 min and the OCT on the tissue was washed with distilled water; The second step was to immerse in 60% isopropanol for 2 minutes, add 200 μL of Oil Red working solution and incubate at room temperature in the dark for 10 minutes; Step 3: Soak in 60% isopropyl alcohol twice for 1 minute each time, then rinse with water immediately after cleaning; Step 4: Use hematoxylin to stain for 20 seconds, run water to reverse blue for 2 minutes, and absorb excess water with absorbent paper; Step 5: Seal the slides with glycerol-gelatin sealing solution, observe under an upright microscope, and take photos.

[0055] Reference Figure 6 Oil red O staining results showed that there were a large number of dense orange-red lipid droplets in the model group mice, indicating that ethanol exposure led to severe lipid accumulation. Both the high-dose group and the silybin group significantly reduced the size and number of hepatic lipid droplets, indicating that mogroside V alleviated lipid accumulation in the liver.

[0056] The above contents are further detailed descriptions of the present invention in combination with specific embodiments, and it cannot be considered that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, making several equivalent substitutions or obvious modifications without departing from the concept of the present invention, and having the same performance or use, should be regarded as belonging to the patent protection scope of the present invention determined by the submitted claims.

Claims

1. Application of Momordica Saponin V in the preparation of alcohol-detoxifying and liver-protecting drugs.

2. The use according to claim 1, characterized in that: The mogroside V reduces alcohol-induced liver inflammation.

3. The use according to claim 1, characterized in that: The mogroside V reduces alcohol-induced liver fat deposition.

4. The use according to any one of claims 1 to 3, characterized in that: Mogroside V is used alone or in combination with Chinese herbal medicines or chemical drugs having the effects of sobering up and protecting the liver.

5. The use according to any one of claims 1 to 3, characterized in that: The mogroside V is combined with a pharmaceutically acceptable carrier to prepare an oral preparation.

6. The use according to claim 5, characterized in that: The oral preparation is a tablet, capsule, granule, spray, lozenge, pill or oral liquid.

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